The Cellular Components of the Adaptive Immune System
The cellular components of the immune system are divided into the cells of the innate immune system and the cells of the adaptive (acquired or specific) immune system. Cells of the innate immune system include (among others) monocytes, granulocytes, natural killer cells in peripheral blood, and mast cells, macrophages and dendritic cells (DCs) in extravascular compartments including peripheral tissues, such as skin, airways, gastrointestinal and urogenital tracts, internal organs as well as secondary lymphoid tissues, such as spleen, lymph nodes (LNs) and Peyer's patches (PPs). The main functions of innate cells are a) provision of immediate protection by neutralizing and limiting dissemination of infectious particles, and by tumor cell clearing, b) immune surveillance of healthy tissues, and c) initiation of adaptive immune responses. Cells of the adaptive immune system include lymphocytes, such as T and B cells. They are distinguished from innate cells by the presence of clonotypic cell surface antigen receptors, referred to as T cell antigen receptor (TCR) and B cell antigen receptor (BCR). Each individual lymphocyte carries a distinct TCR or BCR that recognizes a particular antigen. The specificity of antigen recognition is determined by rearrangement of multiple variable TCR or BCR gene segments during T and B cell development and during antigen affinity maturation at the time of effector T and B cell generation. Naïve, antigen-inexperienced T cells in peripheral blood differ from each other in the antigen-selectivity of their TCRs, and individual naïve T cells become expanded in response to immune activation by agents containing the antigen they are specific for. Consequently, during adaptive immune responses a set of naïve T cells with TCRs specific for the potentially infectious agent becomes expanded via cell proliferation, and develops into a) effector T cells for immediate participation in the defence against the potentially infectious agent, and into b) memory T cells for long-lasting protection against this particular potentially infectious agent. Effector T cells are short-lived, i.e. disappear during the resolution phase of the immune response, whereas the memory T cells are long-lived and are divided into memory T cell subsets according to their primary tissue residence or preferential recirculation routes (Moser et al., 2004). T cells are further divided into αβ T cells and γδ T cells (see below) according to the composition of the heterodimeric TCRs; αβ-TCRs are composed of α- and β-protein chains, and γδ-TCRs are composed of γ- and δ-protein chains. The majority (>80%) of all CD3+ T cells in a normal, healthy person are αβ T cells. TCRs are associated with the invariant CD3 molecule that distinguishes T cells from B cells and all other types of immune cells. The majority of αβ T cells recognize the antigen in a so-called major histocompatibility complex (MHC) molecules-restricted fashion. This is in contrast to BCRs in B cells that directly bind the nominal antigen in a MHC-non-restricted fashion. The term MHC-restriction refers to the mode by which the TCRs recognize their antigens and involves the presentation of antigenic peptides together with MHC molecules, as so-called MHC-peptide complexes, on antigen-presenting cells (APCs), including DCs (see below). There are two major classes of MHC molecules, MHC class I (MHC-I) and MHC class II (MHC-I), which trigger the TCRs of the two major subsets of αβ T cells, the CD8+ αβ T cells and CD4+ αβ T cells. The TCRs on CD4+ αβ T cells recognize MHC-II-peptide complexes whereas the TCRs on CD8+ αβ T cells recognize MHC-I-peptide complexes. In striking contrast, the major subset of γδ T cells in human peripheral blood does not express CD4 or CD8 and its TCRs do not require MHC-restriction for antigen recognition (see below).
γδ T Cells
γδ T cells are a distinct subset of CD3+ T cells featuring TCRs that are encoded by Vγ- and Vδ-gene segments (Morita et al., 2000; Carding and Egan, 2002). They are further divided according to their primary residence in blood or tissues, the protein chain composition of their VγVδ-TCRs and their antigen selectivity: In humans, Vδ1+-TCR chain expressing γδ T cells (Vδ1+ T cells) predominate in epithelial or epithelia-associated/mucosal tissues of the skin, airways, digestive and urogenital tracts, and several internal organs, and constitute a minor fraction (<20%) of γδ T cells in peripheral blood. The TCRs of Vδ1+ T cells recognize lipid antigens presented by MHC-related CD1 molecules. Further, Vδ1+ T cells respond to stress-associated proteins, including MHC-related molecules MICA and MICB, and heat-shock proteins. They are thought to provide a first-line defence against tumors and otherwise stressed cells and, in addition, are thought to contribute to wound healing, tissue repair and autoimmunity. In human peripheral blood of healthy individuals γδ T cells make up 2-10% of total CD3+ T cells, and the majority (>80%) of peripheral blood γδ T cells are Vγ2Vδ2+-TCR chain-expressing γδ T cells (Vγ2Vδ2+ γδ T cells) (Morita et al., 2000; Carding and Egan, 2002).
The TCRs of Vγ2Vδ2+ γδ T cells are highly selective for small non-peptide antigens of mostly microbial origin and do not require antigen presentation by classical MHC molecules, as is typical for peptide-selective αβ T cells (see above). Vγ2Vδ2+ γδ T cell antigens include prenyl-pyrophosphates, such as isopentenyl pyrophosphate (IPP), alkyl-amines and metabolites of a newly discovered isoprenold biosynthesis pathway found in most human microbial pathogens and commensal bacteria [for example, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP); Morita et al., 2000; Eberl et al., 2003)]. Some of these Vγ2Vδ2+ γδ T cell antigens, such as IPP and alkyl amines, are also released by necrotic tissue cells. The homologue of human Vγ2Vδ2+ γδ T cells, carrying homologous VγVδ-TCRs with selectivity for small non-peptide antigens of microbial origin, does also exist in higher primates, such as macaques, but does not exist in rodents, including mice and rabbits. It is not clear why the presence of Vγ2Vδ2+ γδ T cells is limited to higher primates but it could be speculated that they evolved to satisfy the special need for cellular protection against a distinct, species-specific selection of microbes. Vγ2Vδ2+ γδ T cells rapidly expand in response to the model antigen IPP or microbial extracts in vitro during tissue culture of peripheral blood Vγ2Vδ2+ γδ T cells, or in vivo during vaccination experiments in higher primates, such as macaques (Chen and Letvin, 2003). Also, microbial infections in humans are frequently associated with tremendous expansion of peripheral blood Vγ2Vδ2+ γδ T cells, reaching levels as high as >60% of total peripheral blood CD3+ T cells. These findings support the notion that human Vγ2Vδ2+ γδ T cells play an important role in immune processes during microbial infections (Morita et al., 2000; Carding and Egan, 2002; Chen and Letvin, 2003). Their unique selectivity for non-peptide antigens that are commonly found in microbes, including pathogens and commensal bacteria, suggest that the TCRs in Vγ2Vδ2+ γδ T cells fulfil a similar function as toll-like receptors (TLR) that trigger activation and maturation of DCs and other APCs in response to diverse ligands of microbial origin.
γδ T cells contribute to pathogen elimination by rapid secretion of chemokines that initiate the recruitment of cells of the innate immune system and proinflammatory cytokines (TNF-α, IFN-γ) that stimulate antigen-presenting cells and enhance bacterial killing by granulocytes, macrophages and NK cells (Morita et al., 2000; Carding and Egan, 2002; Chen and Letvin, 2003). They also express natural killer cell receptors for killing of infected or neoplastic tissue cells. These findings support the notion that γδ T cells primarily fulfil innate functions, although secretion of pro-inflammatory cytokines, such as TNF-α, is known also to contribute to local adaptive immune responses. On the other hand, evidence for direct involvement of γδ T cells in adaptive immune responses is not clear-cut. For instance, it is reported that CD1-restricted T cells induce maturing in DCs that present CD1-lipid complexes. Also, studies in mice demonstrated a not further explained role for γδ T cells in B cell responses, and human γδ T cells were shown to regulate B cell responses during in vitro co-cultures (Brandes et al., 2003). Finally, studies in macaques demonstrated that Vγ2Vδ2+γδ T cells were able to mount in vivo memory responses to Mycobacterium bovis antigens (Chen and Letvin, 2003). Collectively, these findings provide evidence that γδ T cells are able to interact with cells of the adaptive immune system, such as B cells and DCs. Most of these immunomodulatory functions were attributed to cytokine production by γδ T cells or were left unexplained. Importantly, none of these findings support a role for γδ T cells in antigen presentation.
Lymphocyte function is Intimately related to the lymphocyte migration potential, as defined by the expression of chemokine receptors and adhesion molecules (Moser et al., 2004). Accordingly, αβ T cells are divided into a) naïve T cells expressing the LN-homing chemokine receptor CCR7 but lacking receptors for inflammatory chemokines, b) short-lived effector T cells bearing distinct combinations of chemokine receptors and inducible adhesion molecules that mirror the inflammatory conditions at the site of infection, and c) three subsets of resting, long-lived memory T cells. The distinction of T cell subsets according to their migration potential, i.e. their expression profile of cell surface chemokine receptors and adhesion molecules, correlates well with their state of differentiation and potential function in immune processes. Of note, the “profiling” of chemokine receptors and adhesion molecules is widely used for phenotypic and functional definition of leukocyte subsets, including DCs, and T and B cells (Moser et al., 2004).
The migration properties of human peripheral blood γδ T cells differ strikingly from those of human peripheral blood αβ T cells (Brandes et al., 2003). Most notably, the majority (>80%) of Vγ2Vδ2+ γδ T cells (hereafter referred to as “γδ T cells”) lacks CCR7 and, thus, is excluded from secondary lymphoid tissues, but features an inflammatory migration profile (Brandes et al., 2003). In clear contrast, the majority (>70%) of αβ T cells in peripheral blood express CCR7, which agrees with their continuous recirculation through secondary lymphoid tissues (spleen, LNs, PPs) where they scan APCs for the presence of the appropriate MHC-peptide complexes. In case of ongoing adaptive immune responses, a selection of αβ T cells becomes activated during contact with APCs presenting their cognate antigens and differentiates into CCR7-negative effector cells with inflammatory homing potential. By contrast, peripheral blood γδ T cells feature an inflammatory migration program for their immediate tissue mobilization in response to inflammatory chemokines produced at sites of infection. Upon activation, e.g. in response to microbial extract antigens or defined small non-peptide antigens (such as IPP), the migration profile in γδ T cells rapidly switches from an inflammatory to a LN-homing phenotype, as evidenced by downmodulation of receptors for inflammatory chemokines and induction of CCR7 (Brandes et al., 2003). By contrast to αβ T cells, γδ T cells are relatively rare in LNs, which agrees with their distinct mode of activation that is fully independent of MHC-restricting APCs present at these locations (Brandes et al., 2003). The frequency of γδ T cells is increased in disease-associated LNs (notably in germinal centers), suggesting that γδ T cells may contribute to the initiation of humoral (antibody) responses and possibly other adaptive immune processes.
Collectively, migration characteristics of human γδ T cells and their occasional presence in LNs suggest a role for these cells in the initiation of adaptive immune responses. However, this role is not further defined and there is no evidence that γδ T cells may function as antigen-presenting cells.
A distinguishing feature of γδ T cells is that their TCRs are selective for conserved non-peptide compounds of microbial or tumor cell origin, which are recognized in a MHC-independent fashion. The most relevant ligands appear to be (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), an intermediate of the non-mevalonate pathway in numerous microbes, and isopentenyl pyrophosphate (IPP), the common end product of both the classical and the non-mevalonate pathway (15, 16). The level of peripheral blood Vγ9Vδ2+ T cells in patients suffering from numerous infections, as exemplified by HMB-PP-producing M. tuberculosis and malaria parasites, is highly elevated (15, 16). Vγ9Vδ2+ T cells are also expanded in patents with certain viral infections and show reactivity to tumors (17, 18). Collectively, TCRs in Vγ9Vδ2+ T cells resemble receptors for pathogen-associate molecular patterns found in professional APCs (19), which become rapidly engaged upon pathogen contact. Of note, this particular TCR selectivity is unique to γδ T cells in higher primates, including man, i.e. γδ T cells from standard laboratory animals do not respond to HMB-PP or IPP.
Activated Vγ9Vδ2+ T cells (termed γδ T-APCs) resemble mature dendritic cells (DCs) in their ability to trigger CD4+ αβ T cell responses (21, 22). However, reactivity to a broad range of pathogens, including intracellular bacteria and viruses, as well as tumors suggested a role for γδ T-APCs in induction of pathogen/tumor-specific CD8+ T effector cells. Here we have examined the ability of activated human Vγ9Vδ2+ T cells to cross-present soluble microbial and tumor antigens to autologous CD8+ αβ T cells.
Dendritic Cells (DCs)
DCs form a distinct class of leukocytes, are derived from hematopoietic progenitor cells in the bone marrow, and primarily reside in extravascular sites that include epithelial/mucosal tissues (skin, airways and gastrointestinal/urogenital tracts, among others) and secondary lymphoid tissues (spleen, LNs, PPs) (Banchereau and Steinman, 1998: Steinman et al., 2003; Banchereau et al., 2004). In peripheral blood, DCs or DC precursors make up less than 1% of mononuclear leukocytes. Distinct DC subsets differ in their tissue localization, as exemplified by interstitial DCs that primarily reside in soft tissues bordering epithelia, Langerhans cells (LCs) present in the epidermis and plasmacytoid DCs with homing preferences for LNs. These DC subsets are fully differentiated non-proliferating cells with a limited life-span of several days to several weeks, indicating that they are continuously replaced under steady-state conditions by bone marrow-derived precursors. By contrast, human memory T cells survive for many years and are maintained by means of steady-state (homeostatic) proliferation.
The principal function of tissue-resident DCs is the uptake and processing of local antigens, their relocation via afferent lymphatic vessels to draining LNs and the initiation of antigen-specific adaptive immune responses (Banchereau and Steinman, 1998; Steinman et al., 2003; Banchereau et al., 2004). DCs also induce tolerance when antigens are presented to T cells under tolerogenic conditions, i.e. in the absence of pro-inflammatory T cell co-stimulation. Similarly, antigen-presenting B cells have been shown to induce tolerance (Zhong et al., 1997). Break in the immunological tolerance against self-antigens is thought to be the frequent cause of autoimmune diseases and, thus, tolerogenic DCs presenting self-antigens are essential regulators of immune homeostasis. In healthy peripheral tissues DCs are present in their fully differentiated but “immature” state. Immature DCs express a set of receptors for inflammatory chemokines for quick recruitment to local infection, inflammation or tissue damage. They themselves are poorly immunogenic, i.e. are not capable of inducing primary adaptive immune responses. Instead they are experts in antigen uptake (by means of receptor-mediated endocytic or fluid phase pinocytic mechanisms), antigen processing and peptide loading onto intracellular MHC-I/II molecules and their cell surface presentation. Since immature DCs do not generally express the LN-homing receptor CCR7 it is not known at present how this type of DCs reaches the T cell areas in spleen, LNs and PPs. A multitude of maturation signals, including virus- or bacteria-derived stimuli that trigger toll-like receptors (TLRs), host cell-derived inflammatory mediators (interferon [IFN]-γ, tumor necrosis factor [TNF]-α, interieukin [IL]-1, prostaglandin E2 [PGE2], tissue growth factors, among others), and T cell co-stimulatory molecules (CD40-ligand/CD154), induce DC “maturation” (Banchereau and Steinman, 1998; Steinman et al., 2003; Banchereau et al., 2004). During the early phase of DC maturation, DCs secrete high levels of inflammatory chemokines for augmentation of the inflammatory response via recruitment of additional immature DCs and cells of the innate immune system (monocytes, granulocytes, natural killer cells). Subsequently, the inflammatory migration program is gradually substituted by a LN-homing migration program characterized by substitution of receptors for inflammatory chemokines with CCR7. CCR7 is essential for efficient relocation of sensitized DCs from peripheral tissues to draining LNs in response to the two CCR7-selective chemokines ELC/CCL19 and SLC/CCL21 present on lymphatic vessels and in the T cell area of spleen, LNs and PPs. Thus, CCR7 expression marks mature or maturing DCs. In addition to LN-homing properties, mature DCs feature stable cell surface expression of MHC-I/II-peptide complexes in large numbers as well as diverse co-stimulatory molecules that are required for proper stimulation of naïve (antigen-inexperienced) αβ T cells (Banchereau and Steinman, 1998; Steinman et al., 2003; Banchereau et al., 2004). DCs are also referred to as “professional” APCs because they are capable of stimulating naïve αβ T cells during primary immune responses. Memory (antigen-experienced) T cells have a lower activation threshold and, thus, respond to less stringent stimulatory regimens. The functional duality of DCs that distinguishes between the two states of differentiation, a) immature, antigen-processing DCs in peripheral tissues and b) relocated mature, antigen-presenting and co-stimulating DCs in the tissue-draining LNs, is a hallmark of DC physiology and is tightly linked to local inflammation, infection or tissue damage. Finally, the outcome (quality and quantity) of the adaptive immune response is largely determined by the “mode” of response initiation. DCs are known to “instruct” naïve T cells within the T cell area of LNs and PPs about the type of immune response required for pathogen elimination (Banchereau and Steinman, 1998; Steinman et al., 2003; Banchereau et al., 2004). Accordingly, the inflammatory environment in the tissue directly influences DC maturation and, due to DC relocation, also determines T cell differentiation within draining LNs. Distinct effector fates of naïve T cell differentiation include specialized subsets of T helper cells (IFN-γ/TNF-α-producing type 1 T helper [Th1] cells, IL-4/IL-5/IL-13-producing Th2 cells, among others), regulatory T cells and cytolytic T cells (CTLs). Effector T cells home to sites of inflammation, rapidly mount effector functions (cytokine secretion, lysis/killing of infected/tumor cells) and have a limited life-span. By contrast, memory T cells are the long-lived product of primary immunization and mount superior immune responses against recall antigens.
Immunity to many pathogens and tumors involves major histocompatibility complex class I (MHC I) restricted, cytotoxic CD8+ αβ T cells, which kill affected leukocytes and non-hematopoietic tissue cells (Doherty et al., 2000; Yewdell et al., 2005). Microbes and tumors frequently interfere with antigen processing or presentation and thus inhibit appropriate antigen-presenting cell (APC) function (Alcami, 2003); also, many microbes do not infect APCs. However, dendritic cells (DCs), the prototype professional APCs (4), can take up exogenous material derived from infected cells and tumors and direct these to intracellular compartments with access to the MHC I pathway, a process known as antigen “cross-presentation” (Yewdell et al., 2005; Cresswell et al., 2005; Rock et al., 2005; Villadangos et al., 2007). Such DCs can trigger expansion and differentiation of microbe/tumor-specific CD8+ αβ T cells. Antigen cross-presentation is conventionally thought to be restricted to DCs, such as interstitial DCs and CD8+ DCs in the spleen of mice (den Haan et al., 2000; Stoitzner et al, 2006: Dudziak et al., 2007).
Dendritic Cells in Immunotherapy
DCs are “nature's adjuvant”, i.e. constitute the most expert cellular system for induction of protective immune responses, and, therefore, are being developed for use in human immunotherapy (Fong and Engleman, 2000; Steinman et al., 2003; Schuler et al., 2003; Figdor et al., 2004). Potential applications include cancer therapy, vaccination against pathogens (such as human immunodeficiency virus [HIV]-1 and hepatitis C virus) and treatment of autoimmune diseases. Current DC therapy protocols include:                1. Isolation and purification of DC precursors from patients' blood (bone marrow-derived CD34+ hematopoietic precursors or peripheral blood CD14+ or CD11c+ cells).        2. Generation of DCs during in vitro cell culture.        3. In vitro antigen loading for peptide-presentation by mature DCs.        4. Treatment of patients with single or repeated injections of peptide-presenting DCs.        
Currently, the application of DCs in immunotherapy faces several problems (Fong and Engleman, 2000; Steinman et al., 2003; Schuler et al., 2003: Figdor et al., 2004). In brief, DC precursors are scarce in peripheral blood and do not proliferate during in vitro culture, necessitating repeated manipulation with large blood samples from patients. DCs are functionally heterogeneous and may induce opposing or unwanted effects, e.g. immune suppression instead of effector T cell generation. Also, DCs are functionally instable and go through a preset sequence of irreversible differentiation steps ending in compromised (“exhausted”) immune functions. This causes great difficulties in generating functionally homogeneous DC preparations by in vitro manipulations. Finally, the generation of peptide-presenting DCs for use in immunotherapy is technically demanding, time consuming and costly.